Temperature-Dependent Sample Degradation (Western Blot (Detection Problems))

Question

Accepted Answer

Protein degradation, dephosphorylation, or denaturation occurs when samples are kept above 4°C throughout the protocol, resulting in high background or loss of target bands. Common causes: 1) Lysates or samples not kept on ice during preparation and handling 2) Prolonged exposure to temperatures above 4°C activating proteases and phosphatases 3) Lack of protease inhibitors in lysis buffer Solutions: 1) Ensure all lysates and samples are kept on ice throughout preparation and handling 2) Work quickly and maintain samples at 4°C or below during all protocol steps 3) Add protease inhibitors to lysis buffer; load at least 20–30 µg protein per lane to ensure sufficient target